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1

Strachan, Philip. "Catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259." Thesis, University of Glasgow, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337527.

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2

Harris, Randall. "Degradation of dichloroalkanes by Rhodococcus rhodochrous and Pseudomonas oleovorans." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=98965.

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The degradation of C8-C16 alpha,beta-dichloroalkanes by Rhodococcus rhodochrous ATCC 13808 and Pseudomonas oleovorans ATCC 29347 was studied. The carbon chain length of the alpha,beta-dichloroalkane influenced the degradation rates of these compounds and the observed trends were species-dependent. R. rhodochrous exhibited faster rates towards the longer-chained compounds whereas P. oleovorans most rapidly degraded the shorter-chained compounds. This observation is consistent with the chain-length specificity of the hydroxylase enzymes responsible for initiating the degradation of alkanes by each organism. Studies conducted in a cyclone batch reactor indicated that alpha,beta-dichloroalkanes are modified to chlorinated metabolites prior to the dechlorination step. This indicates that the degradation of alpha,beta-dichloroalkanes is initiated by a hydroxylase acting on the non-chlorinated end of the molecule.
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3

Ashraf, William. "The genetics and biochemistry of a propane-utilizing "Rhodococcus rhodochrous"." Thesis, University of Warwick, 1990. http://wrap.warwick.ac.uk/3991/.

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The pathways of terminal and subterminal propane oxidation have been investigated in a propane-utilizing R. rhodochrous PNKbl. NTG-generated pleiotrophic mutants, characterized by their inability to utilize propane have been isolated. Several classes of mutants have been obtained which are unable to metabolize potential propane oxidation intermediates, e. g. propanol (alcA- or alcB-), propanal (ald-), acetone (ket-), propanoate (oate-) and acetate (ace-). Only ket- mutants retained the ability to metabolize propane. Mutants defective in the first step of propane metabolism (aik-), were also unable to metabolize acetol (a potential subterminal intermediate). Mutant analysis suggests that propane is oxidized via terminal and subterminal pathways in R. rhodochrous PNKbl. However, acetone (a potential subterminal intermediate) does not appear to have a role in propane metabolism. A propane-specific 86 kDa NAD+-dependent secondary alcohol dehydrogenase has been purified to homogeneity. This enzyme oxidizes a range of primary and secondary aliphatic alochols (C2 to C8). It is also responsible for both propan-l-ol and propan-2-ol dehydrogenase activities measured in cell-free extracts of propane-grown cells. Western-blot analysis has shown that it is induced during growth on propane, propan-2-ol, acetol and acetate (subterminal intermediates); but not propan-l-ol, propanal propanoate (terminal intermediates) or acetone. This technique has also demonstrated that a conserved NAD+-dependent alcohol dehydrogenase was induced in Rhodococcus - Nocardia bacteria after growth on propane. SDS-PAGE revealed proteins specific to cells grown on propane and acetol, which may be components of a novel propane/acetol oxygenase system. Oxygenase activity, as demonstrated by the epoxidation of propene, was induced after growth on propane and acetol. NADPHdependent acetol oxygenase activity was also detected. These results suggest a relationship between the metabolism of propane and acetol. Mutants unable to utilize propan-l-ol or propan-2-ol (aicA- and aicB- respectively) were examined by assaying for NAD+-dependent propan-l-ol and propan-2-ol dehydrogenase activities, by using SDS-PAGE analysis of cell-free extracts and comparing the pattern and distribution of pol peptides with the wild-type, and by Western-blot analysis of the NAD -dependent secondary alcohol dehydrogenase synthesized by aicmutants. Results demonstrated the aic- mutants had generally lower NAD+-dependent alcohol dehydrogenase activities altered polypeptide patterns and that alcB mutants synthesized NAD-dependent secondary alcohol dehydrogenase which had altered electrophoretic mobility after non-denaturing PAGE. The latter result may explain the inability of these mutants to utilize propan-2-ol as a growth substrate. The development of a plasmid transformation and gene transfer system for R. rhodochrous PNKbl based on previously published methods has also been assessed. Finally, a model for the pathway of propane oxidation in R. rhodochrous PNKbl is also presented showing oxidation via terminal and subterminal carbon atoms.
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4

Paylor, Michael Mark. "Biotransformation of organosulfides with the bacterium Rhodococcus rhodochrous ATCC 19067." Thesis, University of Exeter, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245300.

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5

Dadd, Michael Richard. "Chiral biotransformations of cylclic nitrile compounds." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365818.

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6

Drago, Gene K. "Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/biology_diss/24.

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Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus
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7

Mihdhir, Alaa. "The physiology, biochemistry and genetics of propane metabolism in Rhodococcus rhodochrous PNKB1." Thesis, University of Warwick, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387396.

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8

Bui, Soi. "Characterisation of the RDX-degrading XplA/XplB redox system from Rhodococcus rhodochrous." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/characterisation-of-the-rdxdegrading-xplaxplb-redox-system-from-rhodococcus-rhodochrous(9355f6f0-62e8-4e07-ad16-786f00a957af).html.

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Hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX) is a military explosive that has become a recalcitrant environmental pollutant over the last few decades owing to its production, storage and use. CYP177A1 (XplA) is a biotechnologically interesting and novel class of P450-flavodoxin fusion enzyme identified from Rhodococcus rhodochrous strain 11Y that catalyses the breakdown of RDX. Its redox partner is a NAD(P)H-dependent FAD-binding flavodoxin reductase (XplB). This study reports the biochemical, biophysical and structural properties of these two enzymes which form a novel P450 redox system with unique domain organisation. These reveal novel features for a P450 enzyme with non-standard UV/Visible spectroscopic features and unusual ligand binding properties. Unexpectedly, XplA’s affinity for imidazole is exceptionally high (Kd = 1.57 μM), explaining previous reports of a red- shifted XplA Soret band in pure enzyme. XplA’s true Soret maximum is at 417 nm. Similarly, the XplA flavodoxin domain displays unusually weak FMN binding (Kd = 1.09 μM), necessitating its reconstitution with the FMN cofactor. Ligand binding data demonstrate XplA’s constricted active site, which can only accommodate RDX and small inhibitory ligands (e.g. 4-phenylimidazole and morpholine) while discriminating against larger azole drugs. The crystal structure identifies a high affinity imidazole binding site, consistent with its low Kd, and shows active site penetration by PEG, perhaps indicative of an evolutionary lipid metabolising function for XplA. The substrate-free heme iron potential (-268 mV vs. NHE) is positive for a low spin P450, consistent with the predominantly reductive role of XplA. The elevated potential of the FMN semiquinone/hydroquinone couple (-172 mV) is also consistent with this functional adaptation. The XplB reductase partner could not be isolated with the FAD cofactor incorporated to make holoprotein. However, the protein was isolated in a soluble and homogenous state which demonstrated very weak FAD affinity. XplB’s ability to interact with XplA and a pyridine nucleotide coenzyme was demonstrated, indicating the enzyme was functional in the presence of FAD. XplA’s unusual molecular selectivity, structural and thermodynamic properties likely reflect its evolution as a specialised RDX reductase catalyst.
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9

Chorao, Charlène. "Etude du métabolisme de Rhodococcus rhodochrous lors de la photobiodégradation du 2-aminobenzothiazole." Phd thesis, Université Blaise Pascal - Clermont-Ferrand II, 2008. http://tel.archives-ouvertes.fr/tel-00731145.

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La biodégradation du 2-aminobenzothiazole (ABT) a été comparée entre des bactéries en suspension dans l'eau et des bactéries immobilisées sur un support d'alginate. Trois processus de dégradation de l'ABT ont été étudiés : la photodégradation sous lumière solaire en présence du complexe Fe(III)-acide nitrilotriacétique (FeNTA), la biodégradation par la bactérie aérobie stricte Rhodococcus rhodochrous et la combinaison de ces deux processus. Le métabolisme de R. rhodochrous a été étudié par RMN in vivo du 31P et du 13C : des informations importantes sur le métabolisme phosphoré et carboné ont été obtenues. La réponse de la bactérie face à divers stress a été évaluée et a montré sa capacité d'adaptation aux variations environnementales. La spéciation du fer pour son rôle important dans l'activation de la biodégradation d'ABT a été etudiée : complexes organiques, oxydes et oxy(hydr)oxydes de fer ont été testés pour connaître les formes biodisponibles pour R. rhodochrous
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10

Zhang, Jie. "Biofiltration of Acrylonitrile by Rhodococcus Rhodochrous DAP 96622 on a Trickling Bed Bioreactor." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/biology_diss/64.

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Acrylonitrile (AN) is a major volatile waste generated in the production of acrylamide and often associated with aromatic contaminants (toluene and styrene) in plant effluents. We examined Rhodococcus rhodochrous DAP 96622 to determine if it could be adapted to efficient biodegradation of acrylonitrile (AN) in a bioreactor. A model bioreactor with granular activated carbon (GAC) as a substratum for Rhodococcus with AN as sole carbon or in combination with toluene was established. The kinetics of AN biodegradation by immobilized and planktonic cells were evaluated and compared. Inlet load and empty retention time were varied to test the removal efficiency in fed-batch and single-pass mode reactor. In addition, the three dimensional structure and characteristics of the biofilm were followed using confocal scanning laser microscopy (CSLM) and relative software. Immobilized cells in the bioreactor, at starting concentrations of AN up to 1150 mg l-1 in the presence of Tol, had at least 13 fold higher AN degradation rates than that seen of planktonic cells. A near steady state of AN degradation was maintained at 75-85% for AN and 80%-90% for Tol within the parameter of EBRT=8 min and AN and Tol inlet loads between 50-200 mg l-1 h-1 and 200-500 mg l-1h-1, respectively. However, when the inlet load of AN was increased to more than 200mg l-1 h-1 and 500 mg l-1 h-1 for Tol, a reduction in efficiency of AN degradation was observed. Biofilms with discrete microcolonies interspersed with voids and channels were observed. Precise measurement of biofilm characteristics agreed with the assumption that the biomass and thickness of the biofilm increased along the carbon column depth. With a porous attachment material like GAC, substrate diffusion is most likely not a limiting factor for AN degradation. Rhodococcus rhodochrous DAP 96622 in a non-sterile activated charcoal column showed efficient degradation of AN in the presence of Tol. The Rhodococcus bioreactor may provide a potential practical waste gas and water treatment system.
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11

Komeda, Hidenobu. "Organization and Regulation of Genes Involved in Nitrile Metabolism in Rhodococcus rhodochrous J1." Kyoto University, 1996. http://hdl.handle.net/2433/160870.

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本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第6519号
農博第895号
新制||農||726(附属図書館)
学位論文||H8||N2948(農学部図書室)
UT51-96-K87
京都大学大学院農学研究科農芸化学専攻
(主査)教授 清水 昌, 教授 駒野 徹, 教授 加藤 暢夫
学位規則第4条第1項該当
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12

Frederick, Joni. "Genetic characterization of Rhodococcus rhodochrous ATCC BAA-870 with emphasis on nitrile hydrolysing enzymes." Doctoral thesis, University of Cape Town, 2013. http://hdl.handle.net/11427/4262.

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Rhodococcus rhodochrous ATCC BAA-870 (BAA-870) had previously been isolated on selective media for enrichment of nitrile hydrolysing bacteria. The organism was found to have a wide substrate range, with activity against aliphatics, aromatics, and aryl aliphatics, and enantioselectivity towards beta substituted nitriles and beta amino nitriles, compounds that have potential applications in the pharmaceutical industry. This makes R. rhodochrous ATCC BAA-870 potentially a versatile biocatalyst for the synthesis of a broad range of compounds with amide and carboxylic acid groups that can be derived from structurally related nitrile precursors. The selectivity of biocatalysts allows for high product yields and better atom economy than nonselective chemical methods of performing this reaction, such as acid or base hydrolysis. In order to apply BAA-870 as a nitrile biocatalyst and to mine the organism for biotechnological uses, the genome was sequenced using Solexa technology and an Illumina Genome Analyzer. The Solexa sequencing output data was analysed using the Solexa Data Analysis Pipeline and a total of 5,643,967 reads, 36-bp in length, were obtained providing 4,273,289 unique sequences. The genome sequence data was assembled using the software Edena, Velvet, and Staden. The best assembly data set was then annotated automatically using dCAS and BASys. Further matepaired sequencing, contracted to the company BaseClear® BV in Leiden, the Netherlands, was performed in order to improve the completeness of the data. The scaffolded Illumina and mate-paired sequences were further assembled and annotated using BASys. BAA-870 has a GC content of 65% and contains 6997 predicted protein-coding sequences (CDS). Of this, 54% encodes previously identified proteins of unknown function. The completed 5.83 Mb genome (with a sequencing coverage of 135 X) was submitted to the NCBI Genome data bank with accession number PRJNA78009. The genome sequence of R. rhodochrous ATCC BAA-870 is the seventh rhodococcal genome to be submitted to the NCBI and the first R. rhodochrous subtype to be sequenced. An analysis of the genome for nitrile
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13

Thuku, Robert Ndoria. "The structure of the nitrilase from Rhodococcus Rhodochrous J1: homology modeling and three-dimensional reconstruction." Thesis, University of the Western Cape, 2006. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3225_1188474860.

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The nitrilases are an important class of industrial enzymes that are found in all phyla. These enzymes are expressed widely in prokaryotes and eukaryotes. Nitrilases convert nitriles to corresponding acids and ammonia. They are used in industry as biocatalysts because of their specificity and enantioselectivity. These enzymes belong to the nitrilase superfamily in which members share a common &alpha
&beta
&beta
&alpha
structural fold and a unique cys, glu,lys catalytic triad with divergent N- and C-terminals.

There are four atomic structures of distant homologues in the superfamily, namely 1ems, 1erz, 1f89 and 1j31. All structures have two-fold symmetry which conserves the &alpha
&beta
&beta
&alpha
-&alpha
&beta
&beta
&alpha
fold across the dimer interface known as the A surface. The construction of a 3D model based on the solved structures revealed the enzyme has two significant insertions in its sequence relative to the solved structures, which possibly correspond to the C surface. In addition there are intermolecular interactions in a region of a conserved helix, called the D surface. These surfaces contribute additional interactions responsible for spiral formation and are absent in the atomic resolution homologues.

The recombinant enzyme from R.rhodochrous J1 was expressed in E. coli BL21 cells and eluted by gel filtration chromatography as an active 480 kDa oligomer and an inactive 80 kDa dimer in the absence of benzonitrile. This contradicts previous observations, which reported the native enzyme exists as an inactive dimer and elutes as a decamer in the presence benzonitrile. Reducing SDS-PAGE showed a subunit atomic mass of ~40 kDa. EM and image analysis revealed single particles of various shapes and sizes, including c-shaped particles, which could not form spirals due to steric hindrances in its C terminal.

Chromatographic re-elution of an active fraction of 1-month old J1 nitrilase enabled us to identify an active form with a mass greater than 1.5 MDa. Reducing SDS-PAGE, N-terminal sequencing and mass spectroscopy showed the molecular weight was ~36.5 kDa as result of specific proteolysis in its C terminal. EM revealed the enzyme forms regular long fibres. Micrographs (109) were recorded on film using a JEOL 1200EXII operating at 120 kV at 50K magnification. Two independent 3D reconstructions were generated using the IHRSR algorithm executed in SPIDER. These converged to the same structure and the resolution using the FSC 0.5 criterion was 1.7 nm.

The helix structure has a diameter of 13nm with ~5 dimers per turn in a pitch of 77.23 Å
. Homology modeling and subsequent fitting into the EM map has revealed the helix is built primarily from dimers, which interact via the C and D surfaces. The residues, which potentially interact across the D surface, have been identified and these confer stability to the helix. The conservation of the insertions and the possibility of salt bridge formation on the D surface suggest that spiral formation is common among microbial nitrilases. Furthermore, the presence of the C terminal domain in J1 nitrilase creates a steric hindrance that prevents spiral formation. When this is lost &ndash
either by specific proteolysis or autolysis - an active helix is formed.

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14

Alves, Marileide Moraes. "Concep??o e estudo de um biofiltro para tratamento de compostos org?nicos vol?teis COVs." Universidade Federal do Rio Grande do Norte, 2005. http://repositorio.ufrn.br:8080/jspui/handle/123456789/15940.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
In the last decade, biological purification of gaseous waste has become an important alternative to many conventional methods of exhaust air treatment. More recently, biofiltration has proved to be an effective and inexpensive method for the treatment of air contaminated with volatile organic compounds (VOCs). A biofilter consists in a reactor packed with a porous solid bed material, where the microorganisms are fixed. During the biofiltration process, polluted air is transported through the biofilter medium where the contaminant is degraded. Within the biofilm, the pollutants in the waste gases are energy and carbon sources for microbial metabolism and are transformed into CO2, water and biomass. The bed material should be characterized by satisfactory mechanical and physical properties as structure, void fraction, specific area and flow resistance. The aim of this research was the biofilter construction and study of the biological degradation of ethanol and toluene, as well as the modeling of the process. Luffa cylindrica is a brazilian fiber that was used as the filtering material of the present work. The parameters and conditions studied were: composition of nutrients solution; effect of microflorae strains, namely Pseudomanas putida and Rhodococcus rhodochrous; waste gas composition; air flow rate; and inlet load of VOCs. The biofilter operated in diffusion regime and the best results for remotion capacity were obtained when a microorganisms consortion of Pseudomanas putida and Rhodococcus rhodochrous,were used, with a gas flow rate of 1 m3.h-1 and molar ratio nitrogene/phosphore N/P=2 in the nutrients solution. The maximum remotion capacity for ethanol was around 90 g.m-3.h-1 and 50 g.m-3.h-1 to toluene. It was proved that toluene has inhibitory effect on the ethanol remotion When the two VOCs were present in the same waste gas, there was a decrease of 40% in ethanol remotion capacity. Luffa cylindrica does not present considerable pressure drop. Ottengraf and van Lith models were used to represent the results obtained for ethanol and toluene, respectively. The application of the transient model indicated a satisfactory approximation between the experimental results obtained for ethanol and toluene vapors biofiltration and the ones predicted it
Na ultima d?cada, a purifica??o biol?gica de rejeitos gasosos tem sido uma importante alternativa em detrimento dos m?todos convencionais de tratamento. Mais recentemente, a biofiltra??o tem se mostrado um m?todo efetivo e de baixo custo para tratamento de ar contaminado com compostos org?nicos vol?teis (COVs). O biofiltro ? um reator que possui um leito filtrante constitu?do por um material poroso, onde est?o fixados os microrganismos. Durante o processo de biofiltra??o, o ar polu?do ? transportado ao longo do leito onde o contaminante ? degradado. No biofilme, os poluentes presentes no efluente gasoso ser?o a fonte de carbono e energia para os microrganismos e ap?s serem metabolizados ser?o transformados em CO2, ?gua e biomassa. O leito filtrante deve ser caracterizado por propriedades f?sicas e mec?nicas como estrutura, fra??o de vazios, ?rea especifica e resist?ncia ao fluxo gasoso. O principal objetivo deste trabalho de pesquisa foi a constru??o de um biofiltro e o estudo da degrada??o do etanol e tolueno, bem como a modelagem do processo. Luffa cylindrica ? uma fibra bastante encontrada no nordeste do Brasil e foi utilizada no presente trabalho como leito filtrante. Os par?metros e condi??es estudos foram: composi??o da solu??o nutriente; efeito da esp?cie de microrganismos, denominadas Pseudomanas putida e Rhodococcus rhodochrous; composi??o do efluente gasoso; taxa de fluxo gasoso e carga de entrada do COV. O biofiltro operou em regime difusional, sendo os melhores resultados para a capacidade de remo??o foram obtidos quando utilizado o cons?rcio de microrganismos de Pseudomanas putida e de Rhodococcus rhodochrous, com uma vaz?o volum?trica de ar de 1 m3.h-1 e raz?o molar nitrog?nio/fosforo N/P=2 na solu??o nutriente. A m?xima capacidade de elimina??o foi de 90 g.m-3.h-1 para o etanol e 50 g.m-3.h-1 para o tolueno. Constatou-se que o tolueno tem efeito inibit?rio sobre a degrada??o do etanol, quando os dois VOCs est?o presentes no mesmo efluente, houve uma queda de 40% na remo??o do etanol. A Luffa cylindrica n?o apresentou grandes valores de perda de carga. Os modelos de Ottengraf e van Lith representaram bem os resultados obtidos para o etanol e tolueno, respectivamente. A aplica??o do modelo transiente evidenciou uma aproxima??o satisfat?ria entre os resultados experimentais obtidos para a biofiltra??o de vapores de etanol e tolueno e aqueles previstos pelo referido modelo
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15

WANG, CUI. "Enhanced Activity And Stability Of Enzymes Associated With Delayed Fruit Ripening In Rhodococcus rhodochrous DAP 96253." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/biology_diss/131.

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Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea). This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions. Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst. The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp. Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).
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Du, Fengkun. "Comparison of Nitrile Hydratases in Rhodococcus Rhodochrous DAP 96253 and DAP 96622 Growing on Inducing and Non-Inducing Media." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/biology_diss/130.

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Nitrile hydratase activity in Rhodococcus rhodochrous DAP 96253 can be induced with multiple inducers that include urea, cobalt (Co), iron (Fe) and nickel (Ni). When induced with Co/urea, cells of R. rhodochrous DAP 96253 expressed the highest level of nitrile hydratase activity (~200 units/min·mg-cdw) when compared with the other inducers tested. Cells induced with Co had the second highest nitrile hydratase activity (~7 units/min·mg-cdw), whereas in the uninduced cells, nitrile hydratase activity was lower than 1 unit/min·mg-cdw. Similarly in R. rhodochrous DAP 96622, when induced with Co/urea, the nitrile hydratase activity of R. rhodochrous DAP 96622 cells was around 50 units/min·mg-cdw which was the highest of all inducers tested. When induced with Co only, the nitrile hydratase activity of R. rhodochrous DAP 96622 was around 20 units/min·mg-cdw, and the nitrile hydratase activity of R. rhodochrous DAP 96622 uninduced was the same as the nitrile hydratase activity of uninduced R. rhodochrous DAP 96253. When Co/urea induced R. rhodochrous DAP 96253 cell lysate was examined on gradient SDS-PAGE and analyzed by Image Quant TL, the nitrile hydratase bands (both α and β subunits) accounted for more than 55% of the total cytosolic proteins. Whereas in Co/urea induced R. rhodochrous DAP 96622, the nitrile hydratase bands accounted for around 25% of the total cytosolic proteins. According to matrix-assisted laser desorption ionization time-of-flight mass spectrometry results, amidase in R. rhodochrous DAP 96253 was approximately 38 kDa from the nitrilase/cyanide hydratase family and amidase in R. rhodochrous DAP 96622 was 55 kDa from the amidase signature family. In addition, the nitrile hydratase regulation system in both R. rhodochrous DAP 96253 and DAP 96622 strains are different. Moreover, the nitrile hydratase regulation system in R. rhodochrous DAP 96253 is different from R. rhodochrous J1. Purified nitrile hydratase from R. rhodochrous DAP 96253 may form a protein complex with glutamine synthetase, resulting in a nitrile hydratase activity of approximately 1500 units/mg-proteins, and nitrile hydratase from R. rhodochrous DAP 96622 is not a protein complex and results in a nitrile hydratase activity of 950 units/mg-proteins.
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17

Rodrigues, Edmo Montes. "Biodiversidade microbiana da Ilha da Trindade, prospecção genética e aplicações biotecnológicas utilizando micro-organismos autóctones." Universidade Federal de Viçosa, 2016. http://www.locus.ufv.br/handle/123456789/10020.

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Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Esse estudo é iniciado com uma revisão bibliográfica em que aborda a biorremediação de hidrocarbonetos pesados e hidrocarbonetos aromáticos policíclicos (PAHs). Na revisão, são explorados aspectos que incluem a obtenção de micro- organismos com capacidade de degradar hidrocarbonetos, vias metabólicas de biodegradação, as consequências ambientais da contaminação e por fim aborda novos estudos que utilizam estratégias e novas tecnologias de biorremediação possíveis em ambientes aquáticos e solos. A parte experimental de nosso estudo segue a mesma linha de pesquisa desenvolvida durante o mestrado. De maneira preventiva, busca-se avaliar e desenvolver metodologias para biorremediar as adjacências da Ilha da Trindade, ambiente pristino e de valor ecológico, caso ocorram acidentes envolvendo o derramamento de hidrocarbonetos. Inicialmente, objetivamos avaliar o genoma dos isolados bacterianos autóctones da comunidade microbiana existente nas águas marinhas que circundam a Ilha da Trindade, Rhodococcus rhodochrous TRN7 e Nocardia farcinica TRH1, isolados durante o trabalho de mestrado como sendo capazes de degradar uma grande variedade de hidrocarbonetos de petróleo. Nosso objetivo também foi avaliar a estrutura da comunidade microbiana e o catabolismo de fenantreno após contaminação de microcosmos contendo água coletada na mesma região e utilizar micro-organismos autóctones como estratégia de bioaumentação. Por fim, objetivou-se desenvolver um produto biotecnológico baseado em emulsões duplas W/O/W fertilizadas, capaz de promover a bioestimulação sem a perda instantânea de nutrientes em águas oceânicas oligotróficas contaminadas por compostos orgânicos hidrofóbicos. Os genomas de R. rhodochrous TRN7 e N. farcinica TRH1 possuem genes de degradação de compostos alifáticos e aromáticos, sendo que 17 genes envolvidos com a biossíntese de antibióticos foram anotados com o genoma de R. rhodochrous TRN7, enquanto que no genoma de N. farcinica TRH1 foram identificados genes relacionados à degradação de caprolactam, precurssor do nylon, e do pesticida atrazina, o que torna ambos os isolados candidatos a serem empregados tanto em estratégias de bioaumentação quanto em outras áreas biotecnológicas. Após a contaminação de microcosmos com hidrocarbonetos, a comunidade microbiana da água litorânea da Ilha da Trindade passa a ser dominada por Gammaproteobacteria, grupo que compõe mais de 72% das sequências analisadas, enquanto que no tratamento controle houve predomínio de Proteobacteria (95% das sequências). Destaca-se a abundância de representantes do gênero Alteromonas, presentes em todos os tratamentos contaminados, com maior valor de abundância (66% das sequências) no tratamento contendo apenas fenantreno como contaminante. Decorridos 30 dias da contaminação, os valores de diversidade foram reduzidos, havendo domínio de grupos bacterianos reconhecidos pela presença de membros com atividade hidrocarbonoclástica. A capacidade de mineralização de fenantreno pela microbiota autóctone da Ilha da Trindade é reduzida quando outros PAHs e hidrocarbonetos de alto peso molecular estão presentes no ambiente. Em relação às emulsões produzidas, nossos resultados mostraram que elas podem ser utilizadas com o propósito de liberação gradual de nutrientes na fase aquosa dispersa. A emulsão é composta por gotículas que variam em um amplo espectro de tamanho que se desestabilizam temporalmente, resultando na liberação da inorgânicos. Quando utilizadas em hidrocarbonoclásticas, fase aquosa conjunto em microcosmos interna, ou contendo não, água contendo nutrientes com bactérias litorânea da Ilha da Trindade contaminada com petróleo, as emulsões W/O/W fertilizadas proporcionaram aumentos significativos da atividade microbiana. Como conclusões, nosso trabalho mostrou que a contaminação por hidrocarbonetos resulta na perda de diversidade microbiana da comunidade microbiana da água litorânea da Ilha da Trindade, e que um ambiente considerado pristino possui microbiota capaz de degradar hidrocarbonetos. Por fim, conclui-se que emulsões duplas W/O/W fertilizadas são uma alternativa para proporcionar aumento significativo na atividade catabólica de hidrocarbonetos em ambientes marinhos oligotróficos.
This study starts with a literature review approach in heavy hydrocarbon bioremediation and polycyclic aromatic hydrocarbons (PAHs). In the review are explored aspects from obtaining microorganisms capable of degrading hydrocarbons, metabolic biodegradation pathways, environmental consequences of contamination and finally discusses new studies using new strategies and bioremediation technologies in water and soil environments. The experimental part of our study follows the same research line developed during the Masters. Preventively, seeks to assess and develop bioremediation methodologies for Trindade Island shoreline, pristine environment with ecological value, should they occur involving the oil spill. Initially we aimed to evaluate the genome of indigenous bacterial isolates Rhodococcus rhodochrous TRN7 and Nocardia farcinica TRH1, isolated during master’s work as being capable of degrading a wide range of petroleum hydrocarbons. Our aim was also to evaluate the microbial community structure and metabolism of PAHs after contamination in microcosms and use indigenous microorganisms as bioaugmentation strategy. Finally, it aimed to develop a biotechnolocal product based on double emulsion W/O/W fertilized able to promote biostimulation without the instant loss of nutrients in oligotrophic oceanic waters contaminated with hydrophobic organic compounds. The genomes of R. rhodochrous TRN7 and N. farcinica TRH1 presents aliphatic and aromatic compounds degradation genes. Moreover, 17 genes involved in the biosynthesis of antibiotics were recorded in the genome of R. rhodochrous TRN7 while in the genome of N. farcinica TRH1 were identified genes related to the degradation of caprolactam, a nylon precursor, and the pesticide atrazine, which makes the isolates candidates to be used in both strategies bioaugmentation as for other biotechnological activity. After contamination by hydrocarbons, in microcosms, the microbial community of the coastal water from Trindade Island becomes dominated by Gammaproteobacteria, a group that makes up more than 72% of the analyzed sequences, while in the control treatment predominated Proteobacteria (95% of the sequences). Noteworthy is the abundance of representatives of the genus Alteromonas, present in all the contaminated treatments with highest abundance (66% of the sequences) in the treatment containing phenanthrene as only contaminant. After 30 days of contamination, diversity values were reduced, with domain of bacterial groups recognized by the presence of members with hydrocarbonoclastic activity. The phenanthrene mineralization capacity by the indigenous microbiota of the Trindade Island is reduced when other PAHs and high molecular weight hydrocarbons are present in the environment, however, does not cease to occur. With respect to emulsions produced, our results showed that they can be used for the purpose of slow release nutrients in the dispersed aqueous phase. The emulsion comprises droplets ranging in a wide range of size that destabilize temporally, resulting in the release of the internal aqueous phase, containing inorganic nutrients. When used together or not, hydrocarbonoclastic bacteria in microcosms containing coastal water from Trindade Island contaminated with oil, the fertilized W/O/W emulsion provided significant increases in microbial activity. In conclusion, our work has shown that hydrocarbons contamination results in the loss of microbial diversity in marine environments, in addition to state that as environment considered pristine has microbiota capable of degrading hydrocarbons and therefore it becomes viable the use of strategies as biostimulation and bioaugmentation employing indigenous bacterial isolates previously selected. Finally, it is concluded that fertilized W/O/W emulsions are an alternative to provide significant increase in catabolic activity hydrocarbons oligotrophic marine environments.
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18

Teixeira, Liliana Patrícia dos Reis. "Production of sophorolipds with a branched hydrophobic tail." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15310.

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Mestrado em Biotecnologia
Sophorolipids are a type of biosurfactants produced by several microorganisms, including the yeast Starmerella bombicola. They find application mainly due to their emulsifying activity and antimicrobial properties. Sophorolipids produced by Starmerella bombicola are composed by one sophorose molecule and a hydroxylated fatty acid of 16 or 18 carbon atoms. One or two acetylations can occur, one on each glucose, and a lactonization as well. Despite the interesting characteristics of classic sophorolipids produced by the referred yeast, there are indications that sophorolipids with branched a hydrophobic tail have higher activity at lower temperatures. These sophorolipids are produced by Rhodotorula bogoriensis, however the yields obtained with this yeast are low. By producing these sophorolipids in Starmerella bombicola, the yields obtained could be higher. Furthermore, the production of these novel sophorolipids will allow broadening of the biosurfactants applications. This thesis describes two approaches to produce sophorolipids with a branched hydrophobic tail: use of Guerbet alcohols as substrates for the Starmerella bombicola culture, and the introduction of genes that will take care of the in-chain hydroxylation of the sophorolipids. Guerbet alcohols are branched molecules, and are expected to be incorporated in the sophorolipids molecules. The genes to be used are from Elizabethkingia meningoseptica and Rhodococcus rhodochrous and encode for an oleate hydratase, an enzyme responsible for the conversion of oleic acid into 10-hydroxystearic acid.
Soforolípidos são um tipo de biosurfactantes produzidos por vários microorganismos, incluindo a levedura Starmerella bombicola. As suas principais aplicações devem-se à sua sua atividade emulsificante e propriedades antimicrobianas. Soforolípidos produzidos por Starmerella bombicola são compostos por uma molécula de soforose e um ácido gordo hidroxilado de 16 ou 18 átomos de carbono. Apesar das interessantes caraterísticas dos soforolípidos clássicos produzidos pela levedura referida, há indicações de que os soforolípidos com cauda hidrofóbica ramificada têm maior atividade a temperaturas mais elevadas. Estes soforolípidos são produzidos por Rhodotorula bogoriensis, no entanto o rendimento obtido por esta levedura é baixo. Ao produzir estes soforolípidos em Starmerella bombicola, o rendimento obtido poderá ser superior. A produção destes soforolípidos irá também permitir o alargamento das aplicações dos biosurfactantes. Esta tese descreve duas medidas para a produção de soforolípidos com cauda hidrofóbica ramificada: uso de álcoois de Guerbet como substrato para a cultura de Starmerella bombicola, e a introdução de genes que serão responsáveis pela hidroxilação no meio da cadeia hidrofóbica dos soforolípidos. Álcoois de Guerbet são moléculas ramificadas, sendo esperado a sua incorporação nas moléculas de soforolípidos. Os genes usados são provenientes de Elizabethkingia meningoseptica e Rhodococcus rhodochrous, e codificam para uma oleate hidratase, enzima responsável pela conversão do ácido oleico no ácido 10-hidroxistearico.
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19

Costa, Vilma Ara?jo da. "Avalia??o da biodessulfuriza??o de 4-metildibenzotiofeno por Rhodococus rhodochrous (NRRL B-2149)." Universidade Federal do Rio Grande do Norte, 2011. http://repositorio.ufrn.br:8080/jspui/handle/123456789/12966.

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Crude oil has between 0.04 up to 5% of sulphur, the higher the oil the higher the sulphur levels. Sulphur usually gives problems such as corrosion in refinery, and once burnt produces SO2 that goes to atmosphere. This work aim to investigate the capacity of Rhodococcus rhodochrous (NRRL B-2149) to metabolize the model compound 4-methyldibenzotiophene (4-MDBT), to remove the sulphur and transform it in 2-hydroxybiphenyl (2-HBF) and sulphite using the 4S pathway. Kynetic runs were carried out in shaker at 120 rpm and 32?C. Samples were taken every 12h to assay substrate consume as well as cells production using HPLC. Results showed that R. rhodochrous NRRL B-2149 can use the 4S pathway in order to remove sulphur without change the carbon chain of the molecule as well as that cells and 4-MDBT affects the product formation. The production of 2-hydroxybiphenyl has interest for industry once it is a potent biocide. However, evaluation is necessary in order to obtain better results compatible with industry needs
O petr?leo bruto convencional cont?m entre 0,04 a 5% de enxofre, e em geral ?leos mais densos apresentam teores mais elevados. Embora pequena, esta fra??o ? importante e sua presen?a torna-se indesej?vel, tanto por promover a corros?o do equipamento da refinaria, como porque parte dela persiste na composi??o dos derivados, que, ao serem queimados, liberam di?xido de enxofre (SO2) para a atmosfera. Este ? um dos principais poluentes atmosf?ricos, respons?vel pelas chuvas ?cidas. O presente trabalho teve como objetivo avaliar a capacidade da cepa Rhodococcus rhodochrous (NRRL B-2149) em metabolizar o composto modelo 4-metildibenzotiofeno (4-MDBT), remover-lhe o ?tomo de enxofre e convert?-lo em 2-hidr?xibifenil (2-HBF) e sulfito utilizando a via sulf?xido-sulfona-sulfonato-sulfato ( 4S ), sem reduzir significativamente seu poder calor?fico. Para essa avalia??o foi realizado um estudo cin?tico em incubador rotativo sob agita??o de 120 rpm e 32?C. As amostras foram retiradas em intervalos de 12 h para an?lise da concentra??o celular e consumo de substrato e quantificadas por cromatografia l?quida. Os resultados mostraram que a cepa de R. rhodochrous NRRL B-2149 ? capaz de utilizar a Via 4S para remover o enxofre do composto sem contudo, alterar sua cadeia carb?nica e que a concentra??o de c?lulas e de 4-MDBT interferem na forma??o do produto 2-HBF. A produ??o do 2-hidroxibifenil detectada neste estudo ? de grande relev?ncia j? que ele ? um biocida potente de interesse industrial. No entanto, avalia-se condi??es dos ensaios visando obter concentra??es vi?veis para amplia??o industrial
2020-01-01
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20

Saqib, Muzna. "Evaluation of Induced Cells of ​Rhodococcus Rhodochrous​ to Inhibit Fungi​." 2016. http://scholarworks.gsu.edu/biology_hontheses/9.

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Rhodococcus rhodochrous is an aerobic, non- pathogenic ,gram-positive bacterium that is often used in industries as a biocatalyst.R. rhodochrous DAP 96253 is capable of exhibiting contact-independent inhibition of selected fungal pathogens.The use of R. rhodochrous as a potential biocontrol agent against plant and animal fungi was examined.The fungi tested were Botrytis cinerea,Pseudogymnoascus destructans,Aspergillus flavus, Fusarium oxysporum’Cigar Tip’ , Rhizopus stolonifer’D1’ ,and other species isolated from berries.Each species was studied to establish the effect of dose (g/cells) and time of exposure to R. rhodochrous.Antifungal inhibition tests were done with the use of dosing,agar diffusion, frozen fermentation paste and exposed slides.Inhibition was observed with B.cinerea,P.destructans,A.flavus and D1,and reduced sporulation was observed with Cigar Tip. The results varied amongst the type of tests used on each target species.
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21

EMMER, Jiří. "Structural studies of the haloalkane dehalogenase mutant (DhaA12) from \kur{Rhodococcus rhodochrous}." Master's thesis, 2007. http://www.nusl.cz/ntk/nusl-46966.

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22

Frederick, Joni. "Characterisation of the nitrile biocatalytic activity of rhodococcus Rhodochrous ATCC BAA-870." Thesis, 2007. http://hdl.handle.net/10539/2016.

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Student Number : 0009756Y - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science
A versatile nitrile-degrading bacterium was isolated through enrichment culturing of soil samples from Johannesburg, South Africa. It was identified as Rhodococcus rhodochrous and submitted to the ATCC culture collection as strain BAA-870. This organism was determined to be a potential biocatalyst in that it contains a two enzyme system with strong nitrile-converting activity comprising nitrile hydratase and amidase. The development of a suitable assay for measuring the activity of the enzymes of interest was explored. A pHsensitive indicator-based assay was found to be suitable only for colorimetrically identifying highly concentrated enzymes with acid-forming activity. An ophthaldialdehyde- based fluorimetric assay was found to be applicable to conversions of select compounds, but the assay could not be used to measure the activity of Rhodoccocus rhodochrous ATCC BAA-870. High performance liquid chromatography was the most suitable method for reliable and quantitative measurement of nitrile hydrolysis, and is applicable to monitoring activities of whole-cell and cell-free extracts. Initial analysis of six compounds, benzonitrile, benzamide, benzoic acid, hydrocinnamonitrile, 3-hydroxy-3- phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid, was performed by HPLC to measure linearly the average retention area, amount and absorbance of the compounds up to 10 mM concentrations. The conversion of the substrates benzonitrile, benzamide and 3- hydroxy-3-phenylpropionitrile were further analysed with respect to time and enzyme concentration. Conversion of benzonitrile to benzamide by the nitrile hydratase was rapid and could be measured in 10 minutes. Conversion of benzamide to benzoic acid by the amidase was considered the rate-limiting step and could be followed for 90 minutes of the reaction at the concentrations tested. Conversion of 3-hydroxy-3-phenylpropionitrile was linearly measured over 20 minutes. Mass spectral analysis was used to confirm, at a structural level, relatively less volatile reactant compounds with a higher thermal stability, including benzamide, 3-hydroxy-3-phenylpropionitrile and 3-hydroxy-3-phenylpropionic acid. Protein concentration studies indicated that activity against benzonitrile was probably due to a nitrile hydratase with potent activity rather than a concentrated enzyme, since formation of benzamide from benzonitrile showed first order reaction kinetics at protein concentrations less than 0.2 mg/ml. Formation of benzoic acid from benzamide was linear up to 1.3 mg total protein and product formation from 3-hydroxy-3-phenylpropionitrile was linear up to 1.4 mg total protein. Overlapping activities against benzonitrile and 3- hydroxy-3-phenylpropionitrile indicate that the nitrile hydratase has differing substrate specificity for the two compounds, with higher activity toward the small aromatic mononitrile, benzonitrile, than the arylaliphatic b-hydroxy nitrile, 3-hydroxy-3- phenylpropionitrile. The nitrile-converting activity of Rhodococcus rhodochrous ATCC BAA-870 would be suitable for biocatalysis as the conversions take place under a wide pH range, require low concentrations of enzyme and reactions are fast. Separation of nitrileconverting activities in Rhodococcus rhodochrous ATCC BAA-870 was undertaken using various chromatography methods to establish a simple, one-step protocol for biocatalytic enzyme preparations. HPLC was not suited to assaying nitrile-converting activity in chromatofocusing fractions, and chromatofocusing Ampholyte buffers were found to interfere with activity measurements. Gel exclusion chromatography of the soluble protein extract from Rhodococcus rhodochrous ATCC BAA-870 indicated the enzyme/s responsible for nitrile hydratase activity are high molecular weight proteins ranging from 40 to 700 kDa in size, while the amidase native enzyme is proposed to be roughly 17 to 25 kDa. SDS-PAGE analysis of gel exclusion and ion exchange chromatography fractions indicated nitrile converting activity in Rhodococcus rhodochrous ATCC BAA-870 is likely due to multimer-forming enzymes made up of 84, 56, 48 and 21 kDa subunits. It is postulated that nitrile hydratase is made up of ab and a2b2 tetramers that may form larger enzyme aggregates. Ion exchange chromatography was used to separate nitrile hydratase with high activity against benzonitrile and 3-hydroxy-3-phenylpropionitrile from amidase activity, and showed that an additional, substrate specific nitrile hydratase may exist in the organism.
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23

Mashweu, Adelaide. "Substrate evaluation of the nitrile degrading enzymes from Rhodococcus rhodochrous ATCC BAA 870." Thesis, 2020. https://hdl.handle.net/10539/31394.

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A dissertation submitted to the Faculty of Science, University of the Witwatersrand in fulfilment for the requirements of the degree of Master of Science, 2020
The focus of this research was the nitrile degrading enzymes nitrile hydratases (NHase) and whole cell nitrilases. NHases and whole cell nitrilases have not been as extensively applied in industry as they could be due to a number of reasons, including missing information relating to their substrate scope. Therefore, this research focussed on exploring the activity of both NHase and whole cell nitrilase towards a number of nitrile compounds. The Groebke-Blackburn-Bienayméreaction (GBB) and Suzuki-Miyaura coupling reaction were employed to synthesize aromatic nitrile-bearing imidazo[1,2-a]pyridine and biaryl compounds respectively. These compounds were of varying sizes and the nitrile group was subjected to different electronic effects through incorporation of different substituents. Vinyl nitrile compounds were synthesized using the Morita-Baylis Hillman reaction (MBH), while simple nitrile compounds were purchased. The synthesized nitrile-bearing imidazo[1,2-a]pyridine compounds were subjected to both NHase and whole cell nitrilase, however, these enzymes were inactive towards these compounds. NHase showed activity towards the biaryl compounds, however, there was complete loss of activity when the biaryl compounds had a 3,4-dimethoxy group as a substituent irrespective of its position relative to the nitrile group. There was also no activity when the 3,4-difluorophenyl group was in the ortho-position relative to the nitrile functional group. NHase also showed activity towards the MBH compounds, however, the rate of hydrolysis was slow in comparison with that of the biaryl compounds. NHase was inactive towards one MBH compound bearing a trimethoxyphenylgroup as a substituent. Whole cell Nitrilase had no activity towards the MBH compounds and extending the time for hydrolysis resulted in no significant changes. NHase demonstrated excellent activity towards the simple commercially available nitrile compounds, however, the rate of hydrolysis towards nitrile compounds bearing electron-withdrawing substituents was faster in comparison with those bearing electron-donating substituents
CK2021
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24

Nunes, Guilherme Duarte Camarinha. "Effect of growth conditions on the activity of Michael hydratase from Rhodococcus rhodochrous ATCC 17895." Master's thesis, 2017. http://hdl.handle.net/10362/25464.

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The stereoselective water addition to double bonds is chemically hard to accomplish. However, the use of a novel Michael hydratase from Rhodococcus rhodochrous ATCC 17895 gives a promising alternative route. The aim of this study is to reveal the dependency of the enzymatic activity on the culture medium and a subsequent optimisation. Focus was given to the composition of the microorganism’s culture medium due to the fact that the literature protocol from 1998 was never changed in this regard. Moreover, the use of iron sulphate and magnesium sulphate in unusually high amounts has drawn the attention to an investigation. By using a “one-variable-at-a-time” (OVAT) strategy, it was proven that the addition of iron sulphate is redundant which enhances the practical treatment of the whole cells. Via a Design of Experiments (DoE) with the support of the software Design Expert v 7.7.0 (Stat Ease, Minneapolis, MN), optimisation of the culture medium parameters’ glucose, yeast extract, peptone and magnesium sulphate was achieved. It was concluded that the addition of magnesium sulphate to the culture medium has no added value. The specific amounts of 6.59 g/L for glucose, 1.84 g/L for yeast extract and 9.20 g/L for peptone provided the best result in a restricted interval of study. Due to this culture medium optimisation, the enzymatic activity was improved by three times compared to the activity provided by the standard culture medium reported in literature. Transferring the shake-flask optimised medium compositions to a fermentor led to large amount of biomass with high Michael hydratase activity thereby saving a substantial amount of growth time. The results of this study provided a significant increase in specific Michael hydratase activity and thereby securing a sufficient amount of active whole-cells needed in the desired Mhy (Michael hydratase) isolation process.
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25

Wang, Cui. "Enhanced Activity And Stability Of Enzymes Associated With Delayed Fruit Ripening In Rhodococcus rhodochrous DAP 96253." 2013. http://scholarworks.gsu.edu/biology_diss/131.

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Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea). This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions. Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst. The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp. Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).
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26

Govindjee, Varsha Parshotam. "Biocatalytic potetial of degrading enzyme systems belonging to the genus rhodococcus rhodochrous, new approaches towards green chemistry." Thesis, 2018. https://hdl.handle.net/10539/25927.

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A Thesis submitted to the Faculty of Chemistry, University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctorate of Philosophy Johannesburg, March 2018
In an attempt to find more efficient and "greener" catalytic processes towards the synthesis of advanced pharmaceutical intermediates, three bacterial strains, namely Rhodococcus rhodochrous ATCC BAA-870, R. rhodochrous A29 and Pimelobacter simplex A99, were evaluated as nitrile hydrolysing biocatalysts. R. rhodochrous ATCC BAA-870 showed constitutive expression of a low-molecular weight cobalt containing nitrile hydratase and a highly enantioselective amidase enzyme that were involved in the kinetic resolution of 3-hydroxy-3-aryloxybutanenitrile and 3-hydroxy-3-arylpropanenitrile. The (R)-3- hydroxy-3-aryloxybutanamide and (S)-3-hydroxy-3-arylpropanamide intermediates were both accumulated with excellent enantioselectivity (>99% ee). Interestingly, the same cells demonstrated enantioselective resolution of β-substituted aminonitriles to amides through a moderately selective nitrile hydratase enzyme when adjusted to a higher pH. With the substrates 3-amino-3-(4-methoxyphenyl)propanenitrile, and 3-amino-3-p-tolylpropanenitrile an enantiomeric purity of the residual nitrile (85% ee and 21% ee respectively) was achieved; while the resultant amide was obtained with 62% ee (3-amino-3-(4-methoxyphenyl)propanamide) and 48% ee (3-amino-3-p-tolylpropanenitrile) respectively. Induction studies demonstrated that both nitrile hydratase and amidase expression in strains A29 and A99 were inducible, with transient expression observed when cultured in the presence of benzonitrile. Furthermore, in a noteworthy discovery, studies on strains ATCC BAA-870, A29 and A99 revealed that the incorporation of 0.5% (v/v) dimethylformamide during cultivation induced nitrilase gene expression. This was evident by the appearance of a new, dominant 40 kDa protein band observed by SDS-PAGE analysis, and confirmed by LCMS-MS sequencing (showing high similarity to Uniprot Q03217). The induced nitrilase in strains A29 and A99 was inhibited by the nitrilase inhibitors benzylamine (61.37% and 79.49%) and benzaldehyde (88.45% and 87.49%) respectively. ATCC BAA-870 cells were however unaffected due to the presence of the alternative nitrile hydratase and amidase system facilitating nitrile hydrolysis. The induced nitrilases converted 3-cyanopyridine to nicotinic acid, and showed excellent enantioselectivity towards 3-amino-3-phenylpropanenitrile (>99% ee), and moderate enantioselectivity towards 3-amino-3-(4-methoxyphenyl)propanenitrile (40% ee). Therefore, Rhodococcal species are capable of expressing various nitrile hydrolysing enzymes that are enantioselective towards classes of beta-substituted nitrile compounds representative of diverse pharmaceuticals such as beta blockers, statins and peptidomimetics. The activity profile of these versatile microbial biocatalyst can be tailored through induction to yield single enantiomer nitriles, carboxamides or carboxyacids.
MT 2018
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27

Jiang, Wenxin. "The Preliminary Study on the Role of 1-Hexene Monooxygenase in Delayed Fruit Ripening by Rhodococcus rhodochrous DAP 96253." 2016. http://scholarworks.gsu.edu/biology_diss/171.

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Abstract:
Rhodococcus rhodochrous DAP 96253, a well-known industrial bacterium, had various 1-hexene monooxygenase (1-HMO) activities when grown on YEMEA plates supplemented with eight different carbohydrates. Besides, 1-HMO exhibited different storage temperature preferences. Lactose could induce the highest 1-HMO activity in R. rhodochrous DAP 96253 while the cells showed the lowest 1-HMO activity when trehalose was the supplement. The 1-HMO activity of R. rhodochrous DAP 96253 was not maintained when stored at 37°C as well as at 4°C and 25°C. Trehalose-induced 1-HMO activity of R. rhodochrous DAP 96253 was more stable from Day 0 to Day 21 at all these three temperatures, compared with the other seven carbohydrates. Immobilization of enzymes can maintain enzyme activity longer, offer easier enzyme storage conditions and make some enzymes reusable, much research has been done in this area. In this study, R. rhodochrous DAP 96253, grown on YEMEA plates supplemented by glucose and urea, was investigated using whole bananas as the inducer of 1-HMO activity and different immobilization methods to maintain this enzyme activity. It was shown that calcium-alginate polyvinyl alcohol (PVA) beads could maintain 1-HMO activity of R. rhodochrous DAP 96253 more stable than calcium-alginate beads. Whole bananas exhibited very obvious effects of inducing 1-HMO activity of R. rhodochrous DAP 96253. A number of recent studies have clearly demonstrated that induced cells of R. rhodochrous DAP 96253 can prolong the shelf-life of post-harvested fruits. With USDA estimates of 40% of all harvested produce in the US not being consumed because of loss of quality, the ability to extend the period of ripeness of produce has great potential to improve the quality of nutrition. Modification or degradation of those signals (primary and secondary) associated with ripening in fruit or the perception of those signals represents a potential mode of action for delayed ripening by induced cells of R. rhodochrous DAP 96253. Ethylene and cyanide are the two primary signals in ripening. In this study, the role of 1-HMO from induced cells was investigated by time-course experiments focusing on 1-HMO activity and stability. In addition, fruit volatile organic compounds (VOCs) were detected and compared by GC-FID and GC/MS over the course of fruit ripening. The results show a correlation between 1-HMO activity and stability in delayed fruit ripening. It was further demonstrated that the presence of secondary signal fruit VOCs enhanced 1-HMO activity. Aromatic profiles of treated fruits, by GC-FID and GC/MS, show a consistent picture of VOCs associated with earlier fruit ripening stages.
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28

Barlament, Courtney. "Process Improvements to Fed-batch Fermentation of Rhodococcus rhodochrous DAP 96253 for the Production of a Practical Fungal Antagonistic Catalyst." 2016. http://scholarworks.gsu.edu/biology_diss/170.

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Abstract:
Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.
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29

STSIAPANAVA, Alena. "Strukturní a funkční studie vybraných mutantů haloalkan dehalogenasy DhaA." Doctoral thesis, 2010. http://www.nusl.cz/ntk/nusl-80498.

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Abstract:
Structural biology is one of the most quickly growing fields of research in life sciences. X-ray diffraction analysis is the technique that allows direct visualization of protein structure at the atomic or near-atomic level. Structure solution of proteins and protein complexes by X-ray crystallography provides important insights into their mode of action. The haloalkane dehalogenase proteins represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. In the present study, the structures of three haloalkane dehalogenase DhaA mutants DhaA04, DhaA14 and DhaA15 at atomic resolution are reported and compared to explore the effect of mutations on the enzymatic activity of modified proteins from a structural perspective. Besides that, in this work, the crystallization and initial X-ray diffraction characterization of DhaA wild type and its mutant variant DhaA13 in complex with environmental pollutant 1,2,3-trichloropropane and the crystallization of DhaA13 in complex with the fluorescence dye coumarin are described.
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